T-cells, including cytotoxic T-lymphocytes, are a critical component of effective human immune responses to tumors, viral infections and other infectious diseases. T-cells destroy neoplastic or virally infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surfaces of target cells. Activation of T-cells is dependent upon coordinate signaling through antigen receptors and costimulator receptors on T-cell surfaces. Antigen presentation, in the absence of simultaneous costimulation, can paradoxically lead to clonal anergy (See, Gimmi et al., Proc. Natl. Acad. Sci. USA, 90:6586 (1993)).
Antigen-presenting cells (APC), such as dendritic cells, are geared towards potent T cell activation, by virtue of their surface MHC and costimulator molecules that can trigger their cognate receptors on specific T cells and thereby provide both critical signals to them (See, Watts et al., Curr. Opin. Immunol., 11:286 (1999); Freedman et al., Cell. Immunol., 137:429 (1991)). Thus, according to the prevalent APC-centric view of T cell regulation, T cell activation is generally viewed as being under the control of professional APC, via intercellular “trans” signaling.
Many mechanisms contribute to the escape of tumor cells and virally infected cells from immune surveillance. One of the mechanisms is that these cells lack the costimulatory molecules required for T-cell activation. “Active” immunotherapeutic strategies have been developed that are predicated upon expressing costimulators on tumor cell, and other antigen-presenting cell, surfaces. An alternative “passive” immunotherapeutic approach involves the steps of recovering tumor-infiltrating lymphocytes (TIL) from tumor beds, or T cells from the blood of cancer patients, expanding the numbers of these T cells ex vivo with lymphokines, and injecting them back into the patient.
A major limiting factor for the clinical application of therapeutic T cells is their loss of activity once injected into patients. This is generally believed to be a consequence of a deprivation of T cell activators, such as soluble cytokines and surface costimulators. Costimulation is required not only for the early activation of T cells, but also for the later maintenance of their post-activation effector capacity, referred to as “effector costimulation.” In an effort to maintain therapeutic T cells in a fully activated state, activating cytokines, such as IL-2, have been administered systemically to patients receiving therapeutic T cells, albeit with IL-2 toxicities and insufficient therapeutic benefit.
T cells also bear inhibitory receptors. The fate of T cells following T cell receptor (TCR) stimulation is guided by the integration of costimulatory and inhibitory receptor inputs. As indicated, costimulatory ligands on APCs trigger cognate receptors on T cells, with resultant enhancement of T cell proliferation, cytokine secretion, and differentiation. In contrast, binding of inhibitory ligand molecules on various cells to cognate receptors on responding T cells diminishes effector functioning, by inducing T cell unresponsiveness or apoptosis.
Professional antigen-presenting cells (APC), by virtue of the surface costimulatory molecules, are geared towards potent T-cell activation. APC can be converted into deletional APC, or “artificial veto cells”, by expressing coinhibitors at their surfaces. This is discussed, for example, in U.S. Pat. Nos. 5,242,687; 5,601,828; and 5,623,056. Such coinhibitors bind to coinhibitor receptors on cells, leading to T-cell inactivation.
One approach for expressing costimulators and coinhibitors on APC, such as tumor cells, is gene transfer. When used for APC and tumor cell engineering, gene transfer techniques have shortcomings. For example, APCs, including tumor cells, are often poorly transfectable. In addition, transfection proceedings are cumbersome and time-consuming. Furthermore, expressing more than a costimulator (or coinhibitor) is difficult. These and other issues have impeded the widespread application of gene therapy for APC and tumor cell engineering.
Protein transfer offers a number of advantages over gene transfer for engineering APCs and other cells. These advantages include the ability to modify poorly transfectable cells (for example, biopsy-derived tumor cells), the simplicity of expressing multiple proteins on the same cell surface, and the relative ease and rapidity of the procedure. The successful use of recombinant GPI-modified costimulator and MHC protein derivatives for protein transfer has been reported. (See, Brunschwig, et al. J. Immunol., 155:5498 (1995); McHugh, et al; Proc. Natl. Acad. Sci. USA, 92:8059 (1995); and McHugh, et al. Cancer Res., 59:2433 (1999)). A shortcoming of the GPI protein transfer strategy, however, resides in scaling up the purification of GPI proteins from membranes of transfected cells. The successful use of protein conjugates consisting of recombinant Fc-modified costimulator derivatives complexed to palmitated protein A for protein transfer has also been reported (See, U.S. Pat. No. 6,316,256; Chen, et al. J. Immunol., 164:705 (2000); Zheng, et al. Cancer Res., 61:8127 (2001)). Yet another protein transfer method entails appending his6-tagged costimulators to cells pre-treated with the chelator lipid, NTA-DTDA, whose NTA groups bind the hexahistidine tags (See, van Broekhoven et al. J. Immunol., 164:2433 (2000)).
Kim and Peacock, J. Immunol. Methods, 158:57 (1993), report the use of palmitate-conjugated protein A for coating cells with artificial receptors which facilitate intercellular interactions. More specifically, a method is reported for attaching an antibody onto the surface of a cell using palmitated protein A. The article does not teach use of a lipidated protein for attachment of anything other than an antibody to a cell. As such, their modified cells serve only as artificial receptors for antigens.
Phillips et al., Immunity, 5:163-172 (August, 1996) report the preparation of a fusion protein using a CD8 leader segment, the Fc domain, of immunoglobin and the ectodomain of a type II membrane protein, CD94. The present transfer methods are applicable to both type I and type II proteins and are neither taught nor suggested in the article.
Darling, et al., Gene Therapy, 4(12):1350-60 (December 1997) report the use of a biotin/avidin-based system for protein transfer. This method involves biotinylation of the target cell, attachment of an avidin group to the protein to be transferred, and combining the biotinylated target cell and the avidin-tagged protein. This method has significant limitations, including its dependence on covalent modifications that could perturb multiple proteins on cell surfaces.
Certain T cell costimulators, including B7-1, B7-2,4-1BB ligand, and OX40 ligand, are expressed on T cells themselves, either under normal conditions or in diseased states (See, Carreno et al. Annu. Rev. Immunol., 20:29 (2002); Nakamura et al. J. Exp. Med., 194:629 (2001); Kochli et al. Immunol. Lett., 65:197 (1999); Wolthers et al. Eur. J. Immunol., 26:1700 (1996); Takasaki et al. Intern. Med., 38:175 (1999); Weintraub et al. J. Immunol., 159:4117 (1997); Weintraub et al. Clin. Immunol., 91:302 (1999)). In addition, it is known that T cells can acquire costimulators from APC via intercellular transfer (See, Hwang et al. J. Exp. Med., 191:1137 (2000); Sabzevari et al. J. Immunol., 166:2505 (2001)), just as they can acquire MHC:peptide antigen complexes via intercellular transfer (See, Lorber et al. J. Immunol., 128:2798 (1982); Hudrisier et al. J. Immunol., 166:3645 (2001)). These costimulators and MHC:peptide antigen complexes on T cells have been presumed to trigger cognate receptors on neighboring T cells in trans.
After T cell triggering, first the inhibitory receptor Fas, and then its cognate ligand, Fas ligand, are sequentially upregulated on T cell surfaces. While some have surmised, on the basis of indirect evidence, the possibility that T cell “suicide” (as a concomitant of T cell “fratricide/sororicide”) might result from such Fas ligand:Fas pairing at the cell surface (See, Brunner et al. Nature, 373:441 (1995); Dhein et al. Nature, 373:438 (1995); Ju et al. Nature, 373:444 (1995)), definitive proof that this mechanism is indeed operative (for example, enforced expression of the ligand:receptor pair) is lacking. Furthermore, while there are other examples of ligand:receptor pairs that are naturally co-expressed on T cell surfaces, for example, CD58 (LFA-3) and its cognate receptor CD2 (See, Springer et al. Annu. Rev. Immunol., 5:223 (1987)), the functional implications of this pairing at the same cell surface (for example, the potential for continuous triggering and/or competitive blockade of incoming trans signals) have been ignored.
The concept of “autocrine signaling”, wherein a cell secretes a soluble protein ligand that binds and signals through one of its own native receptors, has been discussed in the prior literature (See, e.g., Hoffbrand A. V., Semin. Hematol., 30:306 (1993). For example, certain leukemia cells secrete soluble growth factors that can bind to the cell's own receptors, prompting the notion that autocrine signaling results from this ligand:receptor interaction and may play a role in leukemogenesis. However, this literature has dealt exclusively with soluble ligands which bind to the cell's receptors. No therapeutic cells in the art have been designed with membrane-embedded proteins with the capacity to trigger their own activating or inhibitory receptors.
There remains a need, therefore, for methods of efficient and quantitative transfer of proteins and peptides to cells. A further need is to provide such methods in which immunoregulatory or other molecules that retain their function can be attached to cells of interest, including membrane-binding proteins that enable cells to auto-stimulate themselves.